Jul 13, 2023

Public workspaceKarnovsky’s Modified Fixative

DOI
dx.doi.org/10.17504/protocols.io.261genwoog47/v1
  • Mason Mackey1,
  • Mark Ellisman1,2,
  • Marianne Martone1
  • 1National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla CA;
  • 2Department of Neurosciences, University of California, San Diego, La Jolla CA
National Center for Microscopy and Imaging Research
Open access
DOI: dx.doi.org/10.17504/protocols.io.261genwoog47/v1
Protocol Citation: Mason Mackey, Mark Ellisman, Marianne Martone 2023. Karnovsky’s Modified Fixative. protocols.io https://dx.doi.org/10.17504/protocols.io.261genwoog47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 20, 2022
Last Modified: July 13, 2023
Protocol Integer ID: 61106
Keywords: karnovsky, fixative, mice, rat, paraformaldehyde, Glutaraldehyde , cacodylate buffer
Abstract
Fixative for mice and rat tissues to be used during perfusion and immersion fixation.
Guidelines
Preparing Fix solution (2% paraformaldehyde and 2.5% glutaraldehyde)

For 100 mL of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.15M cacodylate buffer solution at 7.4 pH.

Materials
- 100mL Erlenmeyer flask
- 100mL graduated cylinder
- #1 Whatman filter
- Paraformaldehyde PRILLS (Catalog no. 19202)
- Glutaraldehyde EMS (Catalog no. 16220)
- Sodium Cacodylate buffer TED PELLA, INC. (Catalog no. 18851)
- Stir bar
Safety warnings
Attention
Always wear appropriate PPE.
Before start
Make sure heating plate for dissolving paraformaldehyde is between 60-70° C.
Add 2.0 grams paraformaldehyde to a 100mL Erlenmeyer flask.
Add 35mL of DDH2O to 100mL to a Erlenmeyer flask.
Heat paraformaldehyde in DDH2O to 60-70ºC. Use stir bar to mix.
Add 2-3 drops 1N NaOH.
Filter solution with #1 Whatman filter into a 100mL graduated cylinder.
Add 10.0 ml of 25% glutaraldehyde.
Dilute with DDH2O to a total volume 49.0 ml.
Add 1.0 ml of 0.204 M CaCl2-2H2O.
Add 50 ml 0.3M cacodylate buffer stock solution at pH 7.4.
Volume should be 100 ml. Transfer fixative into a 100mL Erlenmeyer flask.
Keep in bath water 35ºC.